The amount of genetic information concerning humans and other species has been expanded enormously, particularly with the advent of the human genome project. With identification of all of the genes present, we will be able to identify mutations associated with particular phenotypes. There is already a substantial library of genes, which when mutated, are known to be associated with various diseases. One need only consider cystic fibrosis, Huntington's disease, .beta.-thalassemia, sickle-cell anemia, and the like. In some instances, such as sickle-cell anemia, there is a common point mutation associated with the disease. In other cases, such as cystic fibrosis, there are numerous point mutations spread throughout the genes associated with the disease.
There are many situations where one would wish to know whether a patient or other species has a point mutation or a particular polymorphism of interest. Not only are we interested in diseases, but particularly with other species, there may be an interest in knowing whether the host has a particular allele.
Numerous techniques have been developed to identify differences between a known and target sequence.
Allele-specific oligonucleotide (ASO) tests are used to identify single-nucleotide mismatches or small differences between a short probe and a target DNA. The target DNA is electrophoresed through a gel and subsequently transferred to a nylon or nitrocellulose membrane. A labelled probe is incubated with the membrane under hybridization conditions which distinguish between the presence and absence of complementarity. The test is dependent upon the strict observance of the hybridization and wash conditions necessary to distinguish between mismatches and complementarity.
The polymerase chain reaction (PCR) has been employed to directly detect sequence differences. One technique known as the amplification refractory mutation system (ARMS) is based on the observation that oligonucleotides which are complementary to a given sequence except for a mismatch of the 3' end will not function as a primer for PCR. Thus, by appropriate selection of primer sets and PCR conditions, one can detect a mismatch. Alternatively, primers may be selected that lead to the formation of normal or mutated amplification products, resulting in a restriction site in one or the other sequence.
Single-stranded conformation polymorphism (SSCP) looks to the detection of single-base differences due to differences in migration rates through non-denaturing polyacrylamide gels (PAGE). After denaturing the target DNA, variations in secondary structure of single-strand DNA can be detected using a non-denaturing gel.
Complementary and mismatched DNA--DNA hybrids denature under different conditions from one another. This has been exploited by denaturing gradient gel electrophoresis (DGGE). DGGE gels contain gradually increasing levels of denaturant causing complementary and mismatched dsDNA molecules to migrate and denature at different points in the gel.
In addition to electrophoresis, there are chemical techniques that may be employed, such as chemical modifying agents that cleave the DNA at the mismatched site, e.g. osmium tetroxide, hydroxylamine, etc.; ribonuclease A cleaves DNA:RNA hybrids at mismatch points; which are then followed by analysis with PAGE. Other techniques include heteroduplex analysis and nucleotide sequence analysis. All of these techniques have limitations in the strictness of the conditions and control which must be employed, the complexity of the protocols, limitations on the generality of the methodology, and the like.
Relevant Literature
Articles which describe various techniques for detecting mismatches include: Dowton and Slaugh, Clin. Chem. 41:785-794 (1995); Newton et al., Nucl. Acids Res. 17:2503-2516 (1989); Haliassos et al., Nucl. Acids Res. 17:3606 (1989); Orita et al., Proc. Natl. Acad. Sci. USA 86:2766-2770 (1989); Sarkar et al., Nucl. Acids. Res. 20:871-878 (1992); Fischer and Lerman, Proc. Natl. Acad. Sci. USA 80:1579-1583 (1983); Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1988); Myers et al., Science 230:1242-1246 (1985); and White et al., Genomics 12:301-306 (1992).